Assessment of canine adrenal function using a compounded synthetic adrenocorticotropic hormone

Diseases of the hypothalamic-pituitary-adrenal (HPA) axis are commonly encountered in dogs and include deficiency or excess of cortisol hormone, termed hypoadrenocorticism and hyperadrenocorticism respectively. Assessment of the HPA axis requires special endocrine assays including the adrenocorticotropic hormone (ACTH) stimulation test, which uses exogenous ACTH to stimulate cortisol production. The most commonly used preparation of ACTH in Australia is Synacthen® (tetracosactrin, Mallinckrodt™ Pharmaceuticals, Staines-upon-Thames, Surrey, UK), which has been intermittently unacquirable. A compounded preparation of synthetic ACTH (tetracosactide, Bova Aus, Caringbah, NSW, Australia) has been available for several years in Australia and offers veterinarians an alternative ACTH for use in testing. It is unknown whether the compounded ACTH has an equivalent effect at increasing blood cortisol concentrations during ACTH stimulation testing compared to Synacthen. Determining its effect in healthy dogs would be the first step towards legitimate use of Bova ACTH in the diagnosis and treatment monitoring of HPA disease in dogs by Australian veterinarians. It is also unknown whether subject variables may affect the HPA axis and ultimately the blood cortisol concentration. - Objectives 1. To establish reference intervals (RI) for an on-site biochemical analyser, as a component of enrolling healthy dogs, prior to its use in the main study. 2. To establish the equivalence of the serum cortisol response to compounded Bova ACTH by comparing serum cortisol concentrations 1 hour after the administration of 5 µg/kg of compounded Bova ACTH versus Synacthen in healthy dogs. 3. To explore the effect of subject factors on the variation of cortisol concentrations in healthy dogs. - Methods RI were established for the biochemical analyser COBAS INTEGRA® 400 plus (Roche, Basel, Switzerland) through a combination of transferring RI from another analyser and generating RI de novo for non-transferable values. The latter used data obtained from assaying stored serum samples from a previous study of healthy dogs. The second objective was achieved through an experimental study of 20 healthy dogs owned by staff and students at a veterinary teaching hospital. A haemogram, biochemistry profile and urinalysis were performed in 20 dogs to confirm their health status prior to enrolment into the ACTH response equivalence study. Ten dogs received Synacthen first and Bova ACTH 24 hours later. Ten dogs received Bova ACTH first and Synacthen 24 hours later. ACTH stimulation testing was performed by collecting blood prior to (t=0), and 1 hour after (t=1) injection of ACTH intravenously. Serum was separated and later assayed for cortisol concentration. Predetermined limits of equivalence were set for the 95% confidence interval (CI) of the ratio of cortisol concentration after stimulation with Bova versus Synacthen to be within 0.8-1.25. Bland Altman analysis provided visual representation of agreement and calculation of mean bias and limits of agreement (equivalence). Passing-Bablok regression analysis was used to examine any systematic and proportional bias between cortisol concentrations after Bova ACTH versus Synacthen stimulation. The final objective was achieved by using the data from the equivalence study to explore subject factors that may explain variance in cortisol concentration after Synacthen stimulation. Variables entered into the multiple linear regression model included neuter status, sex, basal cortisol concentration, age, body condition score, sodium: potassium ratio, haematocrit, neutrophil count, eosinophil count, lymphocyte count, and presence or absence of a stress leukogram. Based on results of this analysis, univariate comparison of serum cortisol concentration for the single-most explanatory variable was performed using a t-test. - Results Seventeen biochemistry analytes had RI established which were then used in screening candidate dogs for ACTH stimulation. Despite normal results from screening haematology, biochemistry and urinalysis, 2 dogs had ACTH stimulation results that were markedly outside of laboratory RI. One dog had very low cortisol concentrations and 1 dog had very high cortisol concentrations. Results from these 2 dogs were removed from further statistical analysis. Of the remaining 18 dogs, the 95% CI of the ratio of cortisol concentration after Bova ACTH/Synacthen was 0.98-1.04. The Bland Altman analysis calculated a bias of 1.01 (95% CI 0.97-1.05) and 95% limits of agreement of 0.87 (95% CI 0.81-0.93) and 1.17 (95% CI 1.09-1.25). Passing-Bablok regression analysis identified no consistent bias. These results confirm that Bova ACTH produces equivalent cortisol concentrations after ACTH stimulation compared to the reference standard Synacthen in healthy dogs. The best single variable explaining the variance in cortisol concentration was neuter status, explaining 48% of the variance in t=1 cortisol concentration. This improved to 55% with the inclusion of basal (t=0) cortisol concentration. Finally, the addition of lymphocyte count improved the explanation to 57%. Entire dogs (3 females and 1 male) had significantly lower mean cortisol concentration compared to neutered dogs (5 females and 9 males). - Conclusion and clinical relevance Bova ACTH results in equivalent cortisol concentrations after ACTH stimulation compared to the reference standard Synacthen in healthy dogs. Bova ACTH is a reasonable alternative for Australian veterinarians when planning ACTH stimulation testing and faced with a shortage of Synacthen, however further research into dogs with HPA dysfunction is needed to fully confirm equivalency of the Bova ACTH preparation. Further research is also needed to determine the role subject characteristics have on the HPA axis, specifically the variation in blood cortisol concentrations. If significant variability is associated with factors such as neuter status, then different RI may be appropriate for dogs that are neutered versus entire.